This protocol compares the relative affinities of binding partners for Rho-family GTPases, including Rac1. In vivo, Rac1-binding proteins compete for a single binding interface, the conformation of which is dictated by a bound nucleotide. The nucleotide is both important and difficult to control experimentally, due to the high hydrolysis rate.
Williamson, R. C., Bass, M. D. Comparing the Affinity of GTPase-binding Proteins using Competition Assays. J. Vis. Exp. (104), e53254, doi:10.3791/53254 (2015).